In biology, a culture is the growing of cells or tissues outside of, and separate from, an organism. Most commonly, and herein, cell and tissue culture refer to the culturing of animal cells, as well as human cultures and cultures from plants, in vitro. Cell or tissue culturing is carried out under precisely controlled conditions, and generally requires the use of a growth medium comprising a specific serum.
Various methods of isolating cells for ex vivo culture are known. These include: purifying white blood cells from blood; breaking down extracellular matrix with enzymes (mononuclear cells); and explant culture, in which pieces of tissue are placed in a growth medium, to grow out cells that are harvested for culture. Cells that are cultured directly from a subject are known as primary cells.
Cells are cultured in an incubator where temperature, humidity, CO2 content, growth medium and light exposure are carefully controlled. These parameters and their optimal values will vary, for various cell types. Some specific parameters that define a growth medium include: glucose concentration, growth factors and other nutrients, pH of the medium. One common growth medium that is suitable for human and mammalian cell types is Dulbecco's Modification of Eagles Medium (a.k.a. Dulbecco/Vogt modified Eagle's minimal essential medium, DMEM). Growth factors derived from blood may be used to supplement the growth medium.
Some cells live without attaching to a surface, and may be cultured in suspension. Cells of the bloodstream are one example. Generally, cells derived from solid tissue must be cultured on a solid substrate to which the cells adhere. Adherent culture cells may be grown on a plastic substrate. The plastic substrate may have a coating that comprises components of the extracellular matrix. Such components increase the adhesion of cells to the substrate and provide other bio-signals needed for growth. Once a viable culture is established, the culture may be further grown and prepared for re-transplantation, experimentation, transfection and transduction, or other purpose.
In a successful cell culture, cells proliferate by division, eventually filling up the available space. Cells in culture may be manipulated to various ends, by various methods. At some point, some cells may be removed from the culture. The particular manipulation depends on whether the culture is in suspension or adherent. For example, suspended cells may be separated from the liquid substrate by centrifuge. In contrast, adherent cells require the breaking of bonds between the cells and proteins that anchor the cells to a solid substrate. Enzymes may be used for this purpose, for example a solution of trypsin-EDTA is commonly used to effect dissociation of anchorage dependent cells from a culture surface.
One challenge faced in tissue engineering research is the ability to stack mono-layers of adherent cells, and maintain the viability of the stack as it grows into a more complex tissue.
Another important challenge faced in tissue engineering research is the ability to produce tissue constructs composed of different types of cells, wherein the constructs are similar to those that assemble naturally in the organism. To achieve this, mono-layers of different cell types would have to be harvested and assembled in a manner that approximates natural tissue growth. It would be convenient to grow mono-layers of various adherent cell types, harvest the layers individually, and then stack the layers one on top of the other, in a well defined manner. Here again, maintaining the viability of the stack of adherent cells until the layered structure can grow into a mature multi-cellular tissue, is a challenge. Thus, new substrate materials and/or a new technique for raising thin layers of adherent cells off of a substrate, are needed.
Taking it a step further, one goal is to regenerate tissues within the patient by delivering prepared cells to a specific site of damage, and then triggering controlled cell growth and differentiation. Achieving this goal will require control over the self-organization of cultured cells into specific arrangements. The implanted cells that proliferate at the site of damage must grow and align themselves precisely, in order to collectively become a functioning tissue. Without meticulous organization, the disoriented cells may interfere with the development of each monolayer of cells and prevent the growth of a comprehensive tissue or organ. Material science has developed biological constructs or scaffolds, that direct and maintain the structure of a tissue. Often, these scaffolds are created with materials that are easier to control than natural biological materials. Nevertheless, there remains a need for improved methods of growing specific arrangements of multi-cell-type tissues, into specific shapes and structures.